Diagnosis and monitoring of liver disease

ABSTRACT

Methods and compositions for screening or diagnosis of liver disease are provided. The results may also indicate course and efficacy of treatment. The composition includes an activity sensor and a reporter releasably attached to the activity sensor. The method includes detecting the presence and/or amount of a reporter. The reporter is released from an activity sensor in the presence of diseased liver tissue but remains attached to the activity sensor in healthy tissue. The presence and/or amount of the reporter are used to characterize the liver disease. The liver disease may be nonalcoholic steatohepatitis (NASH) and results may further indicate staging of NASH.

TECHNICAL FIELD

The present disclosure relates generally to methods and compositions formonitoring physiological state of the liver, such as presence of diseaseor stage.

BACKGROUND

A large number of people suffer from liver diseases and conditions,including non-alcoholic fatty liver disease (NAFLD) and hepatocellularcarcinoma (HCC). HCC is the most common type of primary liver cancer inadults and commonly occurs in people with liver disease. NAFLD is acondition in which excess fat accumulates in the liver of a person whodrinks little or no alcohol. In NAFLD, more than 5-10% of the weight ofthe liver is fat tissue. NAFLD is the most common form of liver diseasein children and has more than doubled over the past twenty years.

When a person suffers from excess fat in the liver, but also suffersfrom inflammation or swelling and liver cell damage, the resultingcondition is known as non-alcoholic steatohepatitis (NASH). Over time,the inflammation and cell damage associated with NASH can lead tofibrosis, or scarring, of the liver. As more and more scar tissue forms,it becomes difficult for the liver to function. When there is long-termdamage of the liver and severe and permanent scarring, the condition isknown as cirrhosis. NASH is a progressive disease that may lead to liverfailure, cancer, or liver transplant. For example, NASH cirrhosis isprojected to be the most frequent reason for liver transplants in theUnited States by 2030. Therefore, accurate, early detection methods areneeded, as NASH may be reversible with early detection.

SUMMARY

The present invention provides an accurate, noninvasive method forcharacterizing a physiological state of liver tissue by detectingactivity of enzymes that are expressed in the liver differentially underthe physiological state of interest. Compositions and methods of thedisclosure are useful for detection and/or staging of liver diseases anddisorders, such as is non-alcoholic fatty liver disease (NAFLD),nonalcoholic steatohepatitis (NASH), hepatocellular carcinoma (HCC),alcoholic liver disease, genetic liver diseases, autoimmune liverdiseases, toxicity-induced liver disease, or metastatic liver cancer. Inparticular, compositions and methods of the disclosure may be used todiagnosis a liver disease and/or to stage fibrosis. The inventioncomprises the use of compositions comprising an activity sensor to whichis attached one or more reporter(s) that is (are) released in the liveronly in the presence of enzymes that are differentially expressed undera physiological condition of interest such as a positive diseasediagnosis or a certain stage of fibrosis. Compositions of the inventionmay be presented to a patient by any acceptable means, including orally,intravenously, sublingually, transdermally, and others. The inventionprovides the ability to screen, diagnose, stage, predict outcome, and/orinform therapeutic choice with respect to disease in, or physiologicalcondition of, the liver. The reporter elements are released from thecore or carrier portion of the activity sensor upon encountering enzymesthat are differentially expressed under a condition of the disease. In apreferred instance, release is accomplished through enzymatic activity,where enzymes that are present at a characteristic abundance (e.g.,differentially over-expressed or under-expressed) in a disease (or at aparticular stage of disease) catalyze the release of reporters that arethen processed or trafficked into a body fluid, ideally urine via thekidneys. Compositions of the disclosure are useful for diagnosing aliver disease such as NASH, NAFLD, or HCC and also for staging acondition such as fibrosis and do so by reporting a particularphysiological state of liver tissue, e.g., by reporting on enzyme(s)that are differentially expressed in the liver tissue under theparticular physiological state.

As discussed below, it is known that the liver accumulates bothendogenous and exogenously-administered materials. In most diagnosticassays, signal from the liver is ignored or is treated as backgroundbecause it is thought that administered compounds accumulate in theliver and create “noise” that obscures or dilutes signal from othersources in the body. The invention makes use of the liver's ability toaccumulate exogenous material and uses that property to advantage inproducing a signal that is highly-specific and sensitive to a number ofdisease states.

The core of the activity sensor can be any molecule that is capable ofaccumulation in the liver and that avoids triggering a significantimmune response. Some detectable but trivial immune response may bepresent upon administration, but is ideally below a predeterminedthreshold. The core must also be capable of carrying or housingreporters for release in the presence of disease. Preferred carriermolecules are discussed below and include proteins, lipids,nanoparticles, and combinations of any of those.

Reporters can be any molecule that is detectable in a body fluid sample,such as urine. In a one aspect, reporters such as peptide fragments arereleased under defined conditions, processed through the kidneys, anddetected in urine. Ideally, reporters are peptides that have a targetsequence for cleavage by a protease. The proteases present in manydisease states in the liver are unique to a particular disease ordisease state (or stage). The invention makes use of the association ofproteases with disease state or stage as a means to cleave reporters,which then will be detected as unique markers for disease. While thereporter can be a peptide that can be sequenced or otherwise detected inurine, it is also contemplated that reports can be non-peptide labelsthat are attached to activity sensors via a linkage, which can be apeptide sequence that is a target of an enzyme. However, release ofreporters from activity sensors can also be accomplished through othermeans, such as light activation, ultrasound, and other means. Reporterscan also be multiplexed in any convenient manner in order to increasespecificity and/or sensitivity. For example, one may wish to includereporters that are linked via peptide recognition sequences for a numberof different proteases or that are recognized by multiple proteases. Forexample, sequences that are cleaved by MMP-9 and MMP-2 may be used inorder to assess for the presence of either of those proteases in thecase in which they are diagnostic of the same or a related condition. Inaddition, multiple reporters designed to assess different disease statescan be used to multiplex the diagnostic/screen.

It is also contemplated that the amount of reporter detected and therate at which the reporter is released is related to disease stage andis informative not only as to diagnosis, but also as to potentialtherapy. One way in which that is done is by using the invention toobtain information on fibrosis staging and rate of progression, whichallows the present invention to be used as a screening along a continuumof liver health. For example, fibrosis staging represents theprogression of fibrosis along the continuum, and is indicated by thelabels F0, F1, F2, F3, and F4. Quantitative measurement of releasedreporters and the rate of released reporters are useful for stagingfibrosis and can be used alone or as an adjunct to traditionaldiagnostics, such as imaging, biopsy, and ultrasound. The presentinvention is also useful to predict clinical efficacy of varioustherapeutic interventions. For example, the rate of fibrosis progressionor regression as determined by methods of the invention is indicative oftherapeutic efficacy.

In general, an activity sensor for use in the invention may be anycarrier that is capable of being administered to a patient and thataccumulates in the liver. A preferred activity sensor comprises an inertcore, an optional linker, and a reporter. The core may be any suitablecore, such as an inorganic molecule, an organic molecule, or a polymer.The core preferably is a polymer composition, such as a polyethyleneglycol (PEG) composition, and the reporter may be derived from a peptidelibrary that may be barcoded in any manner known in the art.

As a non-limiting example of protease promiscuity, MMP9 is an enzymethat may be active to cleave a first sequence in a designed substrate orreporter for an activity sensor. MMP2 may be active for cleaving asecond sequence in a designed substrate. Because the enzymes arecross-react, MMP2 may cleave the first sequence even though it typicallycleaves the second sequence. This means that the substrates designed forparticular protease activity and cleavage in the activity sensors of thepresent invention may be the same because of enzyme promiscuity.

Preferred activity sensors include nanoparticles, inorganic molecules,and organic molecules. The activity sensor may act as a pharmacokineticswitch. For example, one can measure an amount of intact (i.e.,un-cleaved) reporter and an amount of cleaved reporter to assess asignal relating to the presence or absence of disease, staging ofdisease, or the choice of or response to therapy. Generally, a switch isa material having two or more states and that may have differentcirculating activities (e.g., half-life) or dissociation constants.

In a preferred embodiment, activity sensors include polymers (such asPEG) or other vehicles for delivery to the liver. Polyethylene glycol isa preferred carrier (activity sensor), as it typically evades immuneresponse, has a long serum half-life, and is easily accumulated in theliver. Activity sensors may be multiplexed for high specificity anddifferential diagnosis. Released reports are detected in any body fluidsample, but can also be detected in tissue samples obtained from thepatient. A preferred mode of detection is in urine, as released reportermolecules are processed via the kidneys and extracted in urine. Otherpossible bodily fluids for detection include blood, sputum, saliva, andfeces.

Reporters preferably are barcoded for easy detection and associationwith specific enzymes as detailed below. Barcoding can be sequence-basedor can be synthetic markers indicative of a particular reporter or groupof reporters cleaved by a protease or group of proteases. Reporters canbe used to determine stage and rate of disease in a single assay.Additionally, reporters can be multiplexed for detection of multipleenzymes, which aids in the specificity and staging of NASH. Reportersmay also be multiplexed for staging of a disease and rate of progressionof a disease, such as a liver disease (e.g. NASH) or liver cancer (e.g.HCC).

The present invention also contemplates diagnostic methods. Such methodscomprise administering a composition comprising an activity sensor withreleasable reporter molecules to a subject. The composition may beadministered by any suitable manner, e.g. by intravenous injection, byaerosol inhalation, by mouth, or by subcutaneous means. The activitysensor may be part of a timed-release mechanism and may containtargeting agents that promote accumulation in the liver. A sample may becollected after a specified time period, e.g. one hour afteradministration of the composition. The sample may be collected by anysuitable means, e.g. by urine collection. The sample may then beanalyzed, e.g. by mass spectrometry analysis, and the analysis mayfurther use a disease classification algorithm. Results may be issued inthe form of a clinical report that may or may not contain diagnostic orprescriptive information.

Compositions of the invention may include a plurality of carriermolecules, or activity sensors, each having a plurality of reportslinked at a cleavage site. The reports are liberated from the activitysensor or carrier by protease cleavage. Detected reports can be used toproduce a signature that is indicative of disease state and stage. Oneadvantage of the invention is that the proteases active in NASH (whichare indicative of the extent of fibrosis) can be readily determined. Inone example, detection of at least about 10 proteases establishes asignature for NASH at fibrosis stage F2 or greater. RNA sequencing of adiseased sample liver may be used to identify proteases associated withthe liver condition. As a non-limiting example, the proteases selectedmay be from the group consisting of FAP, MMP2, ADAMTS2, FURIN, MMP14,MMP8, MMP11, CTSD, CTSA, MMP12, MMP9, and ST14.

The carrier may be any suitable material, but preferably is apolyethylene glycol (PEG) polymer, such as 40 kDa eight-armpoly(ethylene glycol). Linking peptides to PEG allows for the peptidesto withstand clearance by the kidneys within minutes of administration.Such linking with PEG creates a larger molecular structure and limitsuptake to the cells. PEG is not immunogenic or toxic, thereby allowingfor less frequent administration and lower doses.

Reporters may be made of any suitable material, and preferably are aminoacids, peptides, or polypeptides. The cleavage site is designed to becleaved by specified enzymes, or proteases, indicative of a disease.Although enzymatic cleavage is discussed herein, other methods may beused to cleave reporters. Cleavage by light, pH, ultrasound, andchemical cleavage are non-limiting examples of non-enzymatic activitycleavage.

In designing cleavable reporters, a unique platform was built toengineer nanosensors against protease-mediated diseases. In oneembodiment, 566 proteases across 5 genetic families are identified astargets for activity sensors of the invention. The reporters are linkedin a manner that allows specific cleavage by proteases identified to beinvolved with a disease to be detected. Because the reporters areengineered, the resulting readout may be by any suitable method. Forexample, the readout may be by mass spectroscopy, lateral flow, orELISA.

In another feature of the invention, tissue may be used to identifyproteases active with particular diseases or conditions, which theninforms the design choice of the reporters. As an example, liver tissueis used to identify proteases active in liver disease. A liver samplefrom a patient with a known or suspected disease profile is obtained andRNA is sequenced in order to identify proteases expressed in the knownor suspected disease state. Compositions of the invention are thendesigned so that protease cleavage sites are built into the reporters. Acomposition comprising the carrier and attached reporters is thenadministered to patients and the liberation of protease-specificreporters is determined. It is also contemplated that reporters aredesigned using algorithms based on proteases known to be involved invarious disease states.

In certain aspects, the disclosure provides a method for characterizingliver disease. The method includes detecting a reporter that is releasedfrom an activity sensor differentially in diseased liver tissue versushealthy liver tissue and characterizing a liver disease based upon apresence and/or amount of said reporter. The reporter may be released inhealthy liver tissue, but the activity sensor is designed fordifferential (e.g. greater) cleavage by enzymes that have been shown tobe differentially expressed in liver tissue affected by the disease. Thepresence and/or amount may be determined in a urine sample obtained froma patient to whom the activity sensor was administered (e.g., by massspectrometry). Methods of the disclosure may be used for determining adisease stage and/or for assessing a rate of progression or regressionof a liver disease such as nonalcoholic steatohepatitis (NASH),non-alcoholic fatty liver disease (NAFLD), or hepatocellular carcinoma(HCC).

Methods and compositions of the disclosure may be used for determining adisease stage, and/or may be used for evaluating a physiological stateof tissue or of a subject. For example, methods and compositions of thedisclosure may be used to detect pregnancy, to detect tobacco use, toevaluate diet. In preferred embodiments, methods and compositions of thedisclosure are used to detect, or determine a stage or, a liver disease.Methods may include detecting a plurality of reporters from a pluralityof activity sensors, wherein different ones of the plurality ofreporters are released by enzymes that are differentially active in thediseased liver tissue versus the healthy liver tissue. The enzymesdifferentially active in the diseased liver tissue may include a set ofenzymes wherein activity of the set has been shown to correlate to astage of the liver disease. The enzymes may include, for example, one ormore of a serine protease, a cysteine protease, a threonine protease, anaspartic protease, or a metalloprotease

Methods may include correlating quantities of the plurality of detectedreporters to a rate of progression or regression of the liver disease.In some embodiments, the proteases are selected from the groupconsisting of FAP, MMP2, ADAMTS2, FURIN, MMP14, GZMB, PRSS8, MMP8,ADAM12, CTSS, CTSA, CTSZ, CASP1, ADAMTS12, CTSD, CTSW, MMP11, MMP12,GZMA, MMP23B, MMP7, ST14, MMP9, MMP15, ADAMDEC1, ADAMTS1, GZMK, KLK11,MMP19, PAPPA, CTSE, PCSK5, and PLAU. Preferred embodiments includedetermining a stage of NASH. The activity sensor may include a multi-armpolyethylene glycol (PEG) scaffold and the reporters comprisepolypeptides linked to the PEG scaffold.

In related aspects, the disclosure provides compositions for screeningor diagnosis of liver disease. A composition of the disclosure mayinclude an activity sensor and a reporter releasably attached to theactivity sensor. The reporter is released from the activity sensor inthe liver in the presence of an enzyme that is differentially expressedin liver affected by a disease versus healthy liver. The reporter may bereleased from the activity sensor via enzymatic cleavage. Preferably,the composition includes a plurality of the activity sensors, whereineach activity sensors has a plurality of the reporters releasablyattached thereto, wherein the plurality of activity sensors are releasedby a plurality of enzymes that are differentially expressed in liveraffected by the disease. The plurality of enzymes may be FAP, MMP2,ADAMTS2, FURIN, MMP14, GZMB, PRSS8, MMP8, ADAM12, CTSS, CTSA, CTSZ,CASP1, ADAMTS12, CTSD, CTSW, MMP11, MMP12, GZMA, MMP23B, MMP7, ST14,MMP9, MMP15, ADAMDEC1, ADAMTS1, GZMK, KLK11, MMP19, PAPPA, CTSE, PCSK5,and PLAU (preferably, the composition includes detectable reportersspecific to about eight to twenty or so of those enzymes). In someembodiments, the activity sensor comprises a molecular scaffold and thereporters comprise polypeptides linked to the scaffold. The molecularscaffold may include a polyethylene glycol (PEG) scaffold. The PEGscaffold may include a multi-arm PEG molecule of between about 30 andabout 50 kDa.

In certain embodiments, the activity sensors are designed to query for adisease such as nonalcoholic steatohepatitis (NASH), non-alcoholic fattyliver disease (NAFLD), or hepatocellular carcinoma (HCC). The pluralityof the reporters may each include a polypeptide (or a plurality ofcopies of the polypeptide) that includes a cleavage target of a proteasethat is differentially expressed in liver affected by the disease.Optionally, the plurality of reporters are selected to be cleaved by aset of enzymes that is differentially expressed in a liver affected by acertain stage of NASH. In preferred embodiments, when the composition isinjected into a subject, the plurality of activity sensors collect inthe liver. In an exemplary embodiment, when the plurality of activitysensors collect in a liver affected by NASH stage 2 or higher, theplurality of reporters are cleaved and released from the activitysensors. The cleaved reporters are detectable polypeptides that entercirculation, are filtered from circulation at the kidneys, and areexcreted in urine. Optionally, for mass-spectrometry embodiments, thedetectable polypeptides each has a mass-to-charge ratio that correspondsto an identity of a protease that cleaves that reporter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an embodiment of an activity sensor of the presentinvention.

FIG. 2 illustrates a process of using the present invention.

FIG. 3 shows categories of proteases.

FIG. 4 shows the method for designing a disease-specific nanosensorcocktail.

FIG. 5 shows biological pathways that distinguish NASH from other livertrauma.

FIG. 6 shows participants in NASH pathways.

FIG. 7 gives the results of assaying for proteases that are active inNASH F2+.

FIG. 8 shows the sensitivity and specificity in staging NASH F2+.

FIG. 9 shows detecting NASH at various stages.

FIG. 10 illustrates selection of a subset of proteases.

FIG. 11 shows the sensitivity of sets of proteases.

FIG. 12 shows response of responders vs. non-responders.

FIG. 13 shows organ accumulation of activity sensors.

DETAILED DESCRIPTION

NASH is a disease of protease dysregulation and results in fibrosis,inflammation, and cell death in the liver. NASH is a progressive diseasethat may lead to liver failure, cancer, liver transplant, and death. Themost common liver cancer in adults, typically resulting from liverdisease, is HCC. The conditions associated with NASH and HCC may bereversible with early detection. The present invention provides methodsof detection and staging of liver diseases and conditions, such as NASHand HCC.

The present invention provides non-invasive diagnostic and screeningmethods. However, the present invention may be used in combination withother diagnostic and screening tests. For example, other diagnosticmethods include, but are not limited to, liquid biopsy, tissue biopsy,elastography, biomarker serums, and imaging, such as ultrasound,magnetic resonance imaging (MRI), fluorescence imaging, and positronemission tomography (PET) or computed tomography (CT).

Optionally, the present invention may be used in combination orconjunction with imaging or biopsy. For example, longitudinal monitoringwith the present invention and periodic testing with liquid biopsy orimaging techniques may be performed to assess disease progression and/ortherapeutic selection or efficacy. Other suitable detection methods forliver conditions include, but are not limited to, liver biopsy, liquidbiopsy, ultrasound imaging, elastography, and serum biomarkers, such asthe OWLiver Test from Owl Metabolomics, 13C-methacetin breath test (MBT)from Exalenz Bioscience, Plasma Pro-C3 from Nordic Bioscience, Fibroscanfrom Echosens for transient elastography (TE) using ultrasound, MagneticResonance Elastography (MRE) by Resoundant, Inc., and LiverMultiScanfrom Perspectum Diagnostics.

FIG. 1 illustrates an embodiment of an activity sensor of the presentinvention. The activity sensor 21 comprises a carrier or core 23 withreporter 26 attached thereto. When the carrier 21 is subjected to aprotease 29, the protease 29 cleaves the reporter 26 at a cleavage site25. The liberated reporter is then a detectable analyte 27. In apreferred embodiment, the carrier, or activity sensor, is a polyethyleneglycol (PEG) polymer. For instance, the carrier may be PEG-MAL orPEG-840 kDa. PEG polymers are nontoxic and allow for accumulation in theliver. Use of the PEG carrier provides better bioavailability,circulation time, and safety. Although enzymatic cleavage is discussedherein, other methods may be used to cleave the reporters. Cleavage bylight and chemical cleavage are non-limiting examples of cleavage thatdoes not occur from enzymatic activity.

FIG. 2 illustrates a method 41 of characterizing liver disease accordingto the disclosure. Compositions as described herein may be administered43, for example by intravenous injection, to a subject. Where thecomposition includes the activity sensors 21, the activity sensors willcollect in the liver. In the liver, the cleavage sites 25 are cleaved byproteases active in the liver. An insight of the disclosure is that theset of proteases active together in tissue at any given time provides asensitive marker of disease and the stage of the disease. Also, becausethe activity sensors 21 provide an excess number of substrates for theenzymatic cleavage, the presence of detectable analyte 27 in a samplefrom the body may be measured quantitatively to give a measure of rateof activity of the proteases. Collectively, the rates of activity of theenzymes serve as an instantaneous measure of rate of progression of thedisease. Accordingly, in the method 41, a sample, such as a urinesample, is collected 45. Because the activity sensors 21 are acted uponby the proteases present in the liver, the quantity of the detectableanalytes 27 in the sample provides a measure of stage and rate ofprogression of disease. Thus, the sample is analyzed 47. Any suitableanalysis may be used including, for example, an immune-assay usingantibodies for the detectable analytes 27. In a preferred embodiment,the sample is analyzed 47 by mass spectrometry (which assays for a massto charge ratio of polypeptides cleaved from the activity sensors 21).The output of the analysis 47 is provided to a classifier 49 thatclassifies a stage and/or rate of progression of disease in the tissuebased on the output (e.g., peaks in a mass spectra). The classification49 can be performed by a computer system that uses the classificationresults to provide 51 a report. The report describes a condition of theliver, such as a stage and/or rate of a progression of disease, and maybe used by a physician to consult or treat a patient.

Activity sensors 21 of the disclosure include detectable reporters thatare cleaved and released by enzymatic activity. In preferredembodiments, the reporters 26 of the activity sensors 21 arepolypeptides that include cleavage sites of proteases and are cleaved bythe proteases to release the detectable analytes 27. By includingcleavage sites in the polypeptide reporters 26, the activity sensors 21may be designed to report the activity of any proteases. Any suitableprotease or protease category may be queried by the activity sensors 21,including, for example, cysteine proteases, aspartic proteases, serineproteases, threonine proteases, or metalloproteases.

FIG. 3 summarizes categories of proteases present in human cells acrossthe 5 main enzymatic families. In particular, there are 569 humanproteases, classified as metallo-, cysteine, aspartic, threonine, orserine proteases. Each of those is a target for activity sensors of theinvention. The five main enzymatic families of proteases contribute togrowth, synthesis, signaling, degradation, immunity, and survival incells. Each of those families corresponds to respective catalyticmechanisms. Serine proteases have serine as the nucleophilic amino acidat the active site of the enzyme, cysteine proteases have a catalyticmechanism involving a nucleophilic cysteine thiol, and metalloproteaseshave catalytic mechanisms involving a metal. Similarly, catalyticmechanisms for aspartic proteases involve aspartate residue andcatalytic mechanisms for threonine proteases involve threonine residuewithin the enzymatic active sites, respectively. In the presentinvention, dysregulated proteases that mediate key NASH pathways areused as targets for activity sensors of the invention. The dysregulatedproteases include cathepsins, metalloproteases, trypsins, fibroblastactivation protein (FAP), PLAU, CTSK, MMP-14, PRSS8, PLAT, CASP1, CTSD,ADAM28, GZMA, KLK11, MMP-2, -7, -9, -19, ADAMTS2, FAP, HTRA1, CTSZ, andothers.

FIG. 4 describes how proteases may be selected for inclusion in theactivity sensors 21. A complete set 401, or library, of the activitynanosensors may be designed or made.

For a given liver disease or stage, patient samples may be used tocreate a profile 405 of a level of expression of each protease at thestage of disease. Based on those proteases that are expressedspecifically in the disease condition, specific activity nanosensors 21may be selected from the set 401 for inclusion in a composition 407 ofthe disclosure. The composition 407 preferably include one a pluralityof activity sensors 21 that collectively include reporters 26 that arecleavable by those proteases found to be expressed specifically in thedisease of interest and at a characteristic stage. A specific liverdisease on the liver health continuum considered in the presentinvention is NASH. An insight of the disclosure is that NASH progressesby exploiting certain biological pathways that distinguish NASH fromother liver injury such as cirrhosis. Those pathways involve theexpression and activity of extracellular proteases that promote theprogression of the disease. Moreover, the set of those proteases thatare present and active at each stage of NASH will be characteristic to aspecific stage and can distinguish NASH from other injury by theparticipation of those hallmark NASH pathways.

FIG. 5 shows the four main pathways to NASH. As compared to other liverinjury, NASH is characterized by distinct and specific protease activityin lipogenesis, inflammation, fibrosis, and proliferation pathways. Thepathways are the indicators for NASH and differentiate NASH from otherliver diseases such as cirrhosis or hepatic fibrogenesis. In NASH, theimmune and inflammatory response involves an interaction among theliver, gut, and adipose tissue. Various factors, including metabolicfactors and innate immune alterations, including inflammation cause bybacterial lipopolysaccharide (LPS), fatty acids, chemokines, cytokines,and adipokines, e.g. interleukin-6 (IL-6), tumor necrosis factor α(TNFα), contribute to inflammation in the liver and steatosis. Theactivity sensors provide the ability to diagnose multiple points along apathway. In a preferred aspect of the invention, the pathway is the NASHliver health continuum, which is shown in FIG. 5.

FIG. 6 shows the cells, secreted proteases, membrane-bound proteases,and targets that correspond to the pathways for lipogenesis,inflammation, fibrosis, and cell proliferation. For the lipogenesispathway, FGF21 and IGFBPs are targets, MMP-11 and CTSD (IC) are secretedproteases, FAP and MMP-14 are membrane-bound proteases, and the cellsare hepatocyte, fibroblast, and stellate cells. For example, in theinflammation pathway, CCLs, IL 1b, IL-8, TNFα, LAMP2, Pro-C8, and GSK3βare targets, MMP-2, MMP-11, MMP-9, Furin (IC), CTSD (IC), and CTSA (IC)are secreted proteases, and the cells are hepatocyte, fibroblast,stellate cells, and Kupffer cells. For the fibrosis pathway, I, II, III,IV, Elastin, Fibronectin, Myelin, Integrins, Laminin, Vitronectin, andMMPs are targets, MMP-9, MMP-12, MMP-2, CTSD (IC), and Furin (IC) aresecreted proteases, ST14, FAP, MMP-8, and ADAMTS2 are membrane-boundproteases, and the cells are lymphocyte, neutrophil, Kupffer cells,hepatocyte, fibroblast, and stellate cells. For the cell proliferationpathway, TGFβ, HGF, PDGF-D, FGFs, and VEGF are targets, MMP-2, CTSD(IC), and Furin (IC) are secreted proteases, ST14, FAP, and ADAMTS2 aremembrane-bound proteases, and the cells are hepatocyte, fibroblast, andstellate cells.

FIG. 7 gives the results of assaying for proteases that are active inNASH at stage F2 and above differentially over NASH at stages 0 or 1 orother liver conditions. It is apparent from visual inspection that theproteases MMP9, MMP7, and MMP2, for example, are differentially highlyexpressed in NASH F2-F4 and are understood to be active in the fibrosispathway. Similarly, the protease FAP is highly differentially expressedand is active in the proliferation pathway. Additionally, the proteasePRSS8 is active in NASH F2 and contributes to the inflammation pathway.The protease PLAU is differentially expressed in NASH F2-F4 andcontributes to the lipogenesis pathway. Thus it may be understood thatconstructing an activity sensor 21 with polypeptides as the reporters 26and in which those polypeptides included the specific amino acidsequence that provide cleave sites for MMP9, FAP, MMP7, MMP2, MMP19,ADAMTS2, PRSS8, and PLAU, would provide a composition useful fornon-invasively detecting activity in the liver in which hallmarkproteases of all four biological pathways of NASH are probed todistinguish NASH from health or otherwise injured liver. Additionally,in this example, eight proteases are included. In some embodiments, thedisclosure contemplates an 8-arm PEG scaffold as the carrier, with thereporters 26 linked to the PEG subunits. Using such a composition, thepresent disclosure is useful for diagnosis, staging, monitoring, andtreatment of NASH at any stage, which may be a clinical trial entrycriteria. As such, the present invention is used to identify NASHpatients suitable for treatment.

Additionally, the measurement of the detectable reporters 27 in urinesamples provides for very sensitive and specific detection of the stageand rate of progression of NASH.

FIG. 8 shows the ROC for NASH F2-4 vs. a training set. The axes aresensitivity and specificity. The area under the curve (AUC) was 0.99.

FIG. 9 give the results from detecting NASH at each of a plurality ofstages using compositions of the disclosure. In some embodiment,designing and providing the activity sensors 21 includes testing theactivity sensors 21 in a subject and confirming the results byhistology. By comparing the results of, e.g., multiple replicate trialswith the activity sensors, the sensitivity or specificity of theactivity sensors 21 may be empirically shown. The graphs showreceiver-operator curves (ROC) for stages F0-F4. As shown, the AUC forF0+ was 0.984. For stage F1, the AUC was 0.989. For stage F2+, the AUCwas 0.996. For stage F3+, the AUC was 0.995. For stage F4, the test AUCwas 1.

Preferred embodiments of the disclosure include assaying for proteasesthat are differentially expressed in liver affected by NASH at a givenstage, and methods my further include selecting a subset of thoseproteases to probe via the activity sensors 21. For example, byperforming expression analysis (e.g., by RNA-Seq) on tissue samples fromliver affected by a known stage of NASH, one may identify a large numbere.g., tens, dozens, or more proteases that are active in the tissue. Onemay then select a limited number of those proteases for which it issufficient to probe activity to stage NASH.

FIG. 10 illustrate a set of 34 proteases that are identified asdifferentially expressed in NASH F2+ and then also a set of 10 of thoseproteases that are determined to be informative in classifying NASHstage. The 34 proteases include ADAMTS2, FAP, MMP2, MMP14, PRSS8, GZMK,MMP9, ST14, MMP19, CTSD, CTSW, MMP7, GZMA, FURIN, PCSK5, GZMB, MMP8,MMP23B, PLAU, CTSS, MMP28, CASP1, PAPPA, ADAM12, CTSZ, ADAMTS12,ADAMDEC1, MMP12, CTSA, KLK11, MMP11, CTSE, MMP15, and ADAMTS1. Theinitial 34 proteases are then narrowed to 10. Those included FAP, MMP2,ADAMTS2, MMP14, GZMK, GZMB, FURIN, MMP9, CTSD, and ST14. Tests may beperformed to establish that the subset of a limited number of thedifferentially expressed proteases are useful to classify and stage NASHwith statistical defensibility.

FIG. 11 shows the sensitivity of the full set of 34 proteases inclassifying NASH stage and the sensitivity of the subset of 10 proteasesfor so classifying NASH. For the 34 ranked proteases, the validated AUCwas 0.997. For the 10 ranked proteases, the validated AUC was 0.992. Assuch, it is clear that a high sensitivity and selectivity may beachieved using a subset of all of the proteases that are identified asdifferentially expressed in a stage of NASH.

Aspects of the invention provide a method for characterizing liverdisease. The method includes detecting the presence of a reporter thatis released from an activity sensor in the presence of diseased livertissue but that remains attached to the activity sensor in healthytissue and characterizing a liver disease based upon a presence and/oramount of said reporter. The presence and/or amount may be determined ina urine sample obtained from a patient to whom the activity sensor wasadministered. Preferably, the reporter is released via enzymaticcleavage, e.g., by a protease. The protease may include one or any ofFAP, MMP2, ADAMTS2, FURIN, MMP14, MMP8, MMP11, CTSD, CTSA, MMP12, MMP9,and ST14. In certain embodiments, the liver disease is nonalcoholicsteatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), orhepatocellular carcinoma. Methods may be used for determining a stage ofthe disease, e.g., NASH.

In preferred embodiments, the activity sensor comprises a plurality ofreporters. Each reporter may be associated with a presence of a proteasein the liver. Preferably, the method includes establishing a NASHsignature comprising at least about 10 proteases.

The activity sensor may include polyethylene glycol (PEG), e.g., a 40kDa, multi-arm PEG scaffold to which the reporter polypeptides arelinked.

Related aspects provide a composition for screening or diagnosis ofliver disease. The composition includes an activity sensor with areporter releasably attached to the activity sensor. The reporter isreleased from the activity sensor in the liver only in the presence ofan enzyme associated with a liver condition such as nonalcoholicsteatohepatitis (NASH). Preferably, the activity sensor includes aplurality (e.g., at least four, preferably about eight to about twelve)of reporters that are released by distinct enzymes via enzymaticcleavage in liver affected by the disease. Said enzymatic cleavage maybe directed by a protease, with the reporters being polypeptides inwhich the activity sensor comprises a polyethylene glycol molecule.

Compositions and methods of the disclosure may be used to identify thestage of NASH, or the rate of progression. In preferred embodiments,methods include administering to a patient a composition comprising aplurality of the activity sensors 21 and measuring a quantity of thedetectable analytes 27 in a sample from the patient such as a urinesample. The quantities of the several (preferably about four to aboutfourteen) distinct detectable reporters can show the stage of disease inthe patient's liver, a rate of progression of the disease, or both.Additionally, methods and compositions of the disclosure may be used toprofile a condition of a subject's liver to predict response to atherapy, to monitor treatment or remission, or to screen for inclusionin a clinical trial.

In certain embodiments, methods and compositions of the disclosure areused for drug response monitoring. For example, a phase 2 drug combotrial may be conducted. Patients are randomized to receive Drug 1 withor without Drug 2, or Drug 2 alone. Biopsies from patients are sequencedat the baseline and weeks later. Greater than 80% of proteases ofinterest are significantly changed in responders vs. non-responders(p=0.00036).

FIG. 12 shows the ROC curve for drug responders vs. non-responders.Significantly, in the depicted trial, the AUC is 1. The presentinvention may also be used for detection of conditions other than liverdisease or liver cancer. Organ cancers with a high accumulation of thenanosensors according to the present invention are also considered.

FIG. 13 shows the PEG accumulation (RFU/organ) for C57BL/6 mice havingorgan cancers with a high accumulation of nanosensors according to thepresent invention. The bio-distribution of multi-arm PEG carrier isshown by the PEG accumulation (RFU) for liver, kidney, lungs, spleen,heart, and flank fat.

The present invention may be used for detection of liver conditionsother than NASH, such as liver cancer or HCC. For example, proteasesignatures and nanosensors designed according to the present inventionmay be used to classify hepatocellular carcinoma (HCC). In the presentinvention, the activity sensors may be used to differentiate HCC fromchronic liver disease (CLD). Analysis of TCGA human HCC database providemany samples for all stages of HCC, numerous for stage 1 of HCC, andnumerous for non-tumor. 44 proteases are found to be shared amongetiologies including HBV, HCV, ALD, and NAFLD. HCC-specific proteasesare then identified. An area under the curve for training of the stage 1HCC samples vs the non-tumor sample is 0.984. Validation produces anarea under the curve of 0.948 for a stage 1 HCC sample vs a non-tumorsample. As such, the activity sensors may be used to characterize HCCand differentiate from other CLD with high sensitivity and specificity.The area under the curve increases in value when classifying HCC anddifferentiating from other cancer types (training AUC=1.0 for HCC samplevs. non-tumor or other tumor sample; validation AUC=0.999 for HCC samplevs. non-tumor or other tumor sample).

The present invention may achieve greater sensitivity and specificitythan other available detection techniques. Other detection tests for HCCinclude ultrasound, computed tomography (CT), and magnetic resonanceimaging (MM). Liquid biopsy is also a potential testing method fordetection. Ultrasound has an overall sensitivity of 94% and an early HCCsensitivity of 63%. CT has an overall sensitivity of 76% and an earlyHCC sensitivity of 58%. MRI has an overall sensitivity of 85% and anearly HCC sensitivity of 67%. Wako Diagnostics supplies the AFP-L3, DCPbiomarkers test which has an overall sensitivity of 83%. Glycotestsupplies a Glycoprotein fucosylation test with an overall sensitivity of93% and an early HCC sensitivity of 83-92%. Liquid biopsy may be usedfor HCC, but no sensitivity data is presently available. The methods arediscussed in the following articles, which are incorporated herein:Singal et al. Aliment Pharmacol Ther 2009, 30:37-47; Nam et al. ClinicalGastroenterology and Hepatology 2011, 9:161-167; Hann et al., J MedMicrob Diagn 2014, 3:1; Glycotest 2017 Investor Presentation; Mehta etal. Cancer Epidemiol Biomarkers Prev, 26(5) May 2017; Glycotest PressRelease Jan. 4, 2018; and J. D. Cohen et al., Science10.1126/science.aar3247 (2018).

Example 1

The activity sensors of the invention are designed to assess NASHdisease severity and monitor treatment response. The activity sensorsare used to evaluate associations between hepatic protease geneexpression and fibrosis severity and to determine changes in proteaseexpression according to fibrosis response.

For design of the activity sensors specific to NASH, RNA sequencing(RNAseq) is carried out to determine which proteases are expresseddifferentially in NASH. After determining which proteases are expressedin NASH, activity sensors are designed. In particular, reporters in theactivity sensors are designed to be cleaved by the proteasesdifferentially expressed in NASH liver tissue compared to healthy livertissue.

Compositions of the invention are tested in mouse models for NASH. HumanmRNA sequence (RNA-Seq) data are obtained from The Cancer Genome Atlas(TCGA) and used to identify protease targets for detection of NASH.Using a genetic mouse model of NASH, the expression of multiple targetproteases is validated and a panel of protease-responsive activitysensors is designed. Upon administration, reporters are cleaved anddetected in the urine of the mice using LC-MS/MS. Other aspects andadvantages of the invention will be apparent upon consideration of thefollowing detailed description thereof.

Specifically, RNAseq is performed on RNA extracted from procuredformalin fixed and paraffin embedded (FFPE) liver tissue from between 50and 200 patients with NASH and hepatic fibrosis as well as healthycontrols. RNAseq is also performed on RNA extracted from fresh livertissue obtained at baseline (BL) and weeks later (W) from subjects withNASH at various stages of fibrosis, treated with one or moretherapeutics alone or in combination.

Next, the activity sensors are designed. Protease gene expression iscompared between NASH patients and controls using statistical methods,and the associations between protease gene expression and fibrosis stageare evaluated, as well as evaluation of changes in gene expressionaccording to fibrosis response (≥1-stage improvement), between BL and W.Results from analysis indicated that the expression levels of 9 proteasegenes, including FAP, ADAMTS2, MMP14, and MMP15, from multiple diseasepathways including fibrosis, inflammation, and cell death are increasedin NASH patients versus healthy controls (all P<0.05). The expressionlevels of 18 protease genes are positively correlated with fibrosisstage. Between BL and W, the expression of 7 proteases decreased(P<0.05) in patients with fibrosis response compared withnon-responders. Compared to all genes, decreases in target proteases areenriched in fibrosis responders vs. non-responders.

Those results indicate that the hepatic protease expression in patientswith NASH is correlated to fibrosis stage and treatment response. Thus,proteases involved in fibrosis, inflammation, and cell death areimportant in the progression of NASH.

One of skill in the art would know what peptide segments to include asprotease cleave sites in an activity sensor of the disclosure. One canuse an online tool or publication to identify cleave sites. For example,cleave sites are predicted in the online database PROSPER, described inSong, 2012, PROSPER: An integrated feature-based tool for predictingprotease substrate cleavage sites, PLoSOne 7(11):e50300, incorporated byreference. Reproduced below is a set of exemplary protease substratesfor a variety of significant protease. In the sequences shown below, thevertical bar shows the cleavage site, and forms no part of the sequence.Any of the compositions, structures, methods or activity sensorsdiscussed herein may include, for example, any suitable cleavage targetsincluding, for example, any of the sequences below as cleavage sites, aswell as any further arbitrary polypeptide segment to obtain any desiredmolecular weight. To prevent off-target cleavage, one or any number ofamino acids outside of the cleavage site may be in a mixture of the Dand/or the L form in any quantity.

Aspartic protease HIV-1 retropepsin (A02.001) A02.001:SSTS|SWYS (SEQ ID NO: 1) A02.001: PCIQ|AESE (SEQ ID NO: 2) A02.001:DDEP|IELA (SEQ ID NO: 3) A02.001: VLEQ|VVTS (SEQ ID NO: 4) A02.001:QVVQ|VVLD (SEQ ID NO: 5) Cysteine protease Cathepsin K (C01.036)C01.036: KSIQ|EIQE (SEQ ID NO: 6) C01.036: KDFA|AEVV (SEQ ID NO: 7)C01.036: TSYA|GYIE (SEQ ID NO: 8) C01.036: LKVA|GQDG (SEQ ID NO: 9)C01.036: FCLH|GGLS (SEQ ID NO: 10) Calpain-1 (CO2.001) C02.001:WMDF|GRRS (SEQ ID NO: 11) C02.001: SATA|AVNP (SEQ ID NO: 12) C02.001:RELG|LGRH (SEQ ID NO: 13) Caspase-1 (C14.001) C14.004:DEGD|SLDG (SEQ ID NO: 14) C14.004: DETD|MAKL (SEQ ID NO: 15) C14.004:EECD|AAEG (SEQ ID NO: 16) Caspase-3 (C14.003) C14.003:AEVD|GDDD (SEQ ID NO: 17) C14.003: DRHD|GTSN (SEQ ID NO: 18) C14.003:VEVD|APKS (SEQ ID NO: 19) Caspase-7 (C14.004) C14.004:DQTD|GLGL (SEQ ID NO: 20) C14.004: DSID|SFET (SEQ ID NO: 21) C14.004:DDVD|TKKQ (SEQ ID NO: 22) Caspase-6 (C14.005) C14.005:VEMD|AAPG (SEQ ID NO: 23) C14.005: VSWD|SGGS (SEQ ID NO: 24) C14.005:EETD|GIAY (SEQ ID NO: 25) Caspase-8 (C14.009) C14.003:VETD|KATV (SEQ ID NO: 26) C14.003: GSSD|PLIQ (SEQ ID NO: 27) C14.003:DDAD|YKPK (SEQ ID NO: 28) Metalloprotease Matrix metallopeptidase-2(M10.003) M10.003: HISS|LIKL (SEQ ID NO: 29) M10.003:DPNN|LLND (SEQ ID NO: 30) M10.003: DLSD|LTAA (SEQ ID NO: 31) M10.003:FSAY|IKNS (SEQ ID NO: 32) M10.003: EALP|LLVR (SEQ ID NO: 33)Matrix metallopeptidase-9 (M10.004) M10.004: QQGA|IGSP (SEQ ID NO: 34)M10.004: GPPG|IVIG (SEQ ID NO: 35) M10.004: MDIA|IHHP (SEQ ID NO: 36)M10.004: FFKN|IVTP (SEQ ID NO: 37) M10.004: GPLG|ARGI (SEQ ID NO: 38)Matrix metallopeptidase-3 (M10.005) M10.005: HLGG|AKQV (SEQ ID NO: 39)M10.005: VWAA|EAIS (SEQ ID NO: 40) M10.005: GPLG|ARGI (SEQ ID NO: 41)M10.005: ESGD|YKAT (SEQ ID NO: 42) Matrix metallopeptidase-7 (M10.008)M10.008: VAQD|LNAP (SEQ ID NO: 43) M10.008: SPDA|LQNP (SEQ ID NO: 44)M10.008: PPLK|LMHS (SEQ ID NO: 45) M10.008: GPHL|LVEA (SEQ ID NO: 46)Serine protease Chymotrypsin A (cattle-type) (S01.001) S01.001:VGPN|LHGV (SEQ ID NO: 47) S01.001: GGGN|KIGP (SEQ ID NO: 48)Granzyme B (Homo sapiens-type) (S01.010) S26.010:LSTA|RFVV (SEQ ID NO: 49) S26.010: VTED|VDIN (SEQ ID NO: 50)S26.010: SALA|TTVY (SEQ ID NO: 51) Elastase-2 (S01.131) S01.131:QELI|SNAS (SEQ ID NO: 52) S01.131: QELI|SNAS (SEQ ID NO: 53) S01.131:WELI|SNAS (SEQ ID NO: 54) Cathepsin G (S01.133) S01.133:SGNY|ATVI (SEQ ID NO: 55) S01.133: SIQM|NVAE (SEQ ID NO: 56) S01.133:QQNY|QNSE (SEQ ID NO: 57) Thrombin (S01.217) S01.217:SILR|LAKA (SEQ ID NO: 58) S01.217: KFQR|AITG (SEQ ID NO: 59) S01.217:AEPK|MHKT (SEQ ID NO: 60) S01.217: TIPR|AAIN (SEQ ID NO: 61)Plasmin (S01.233) S01.233: AEFR|HDSG (SEQ ID NO: 62) S01.233:RRKN|IVGG (SEQ ID NO: 63) S01.233: AMSR|MSLS (SEQ ID NO: 64)Glutamyl peptidase I (S01.269) S01.269: PEPE|QLKM (SEQ ID NO: 65)S01.269: QSKE|AIHS (SEQ ID NO: 66) S01.269: KLKE|ASRS (SEQ ID NO: 67)Furin (S08.071) S08.071: RAKR|SPKH (SEQ ID NO: 68) S08.071:RKKR|STSA (SEQ ID NO: 69) Signal peptidase I (S26.001) S26.001:SAMA|ADSN (SEQ ID NO: 70) S26.001: TLLA|NINE (SEQ ID NO: 71)Thylakoidal processing peptidase (S26.008) S01.269:QAEE|TYEN (SEQ ID NO: 72) S01.269: DVID|MSKE (SEQ ID NO: 73)Signalase (animal) 21 KDa component (S26.010) S26.010:EVLA|TPPA (SEQ ID NO: 74) S26.010: APVP|GTAW (SEQ ID NO: 75)

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

What is claimed is:
 1. A method for characterizing liver disease, themethod comprising: detecting a reporter that is released from anactivity sensor differentially in diseased liver tissue versus healthyliver tissue; and characterizing a liver disease based upon a presenceand/or amount of said reporter.
 2. The method of claim 1, wherein saidpresence and/or amount is determined in a urine sample obtained from apatient to whom the activity sensor was administered.
 3. The method ofclaim 1, further comprising detecting a plurality of reporters from aplurality of activity sensors, wherein different ones of the pluralityof reporters are released by enzymes that are differentially active inthe diseased liver tissue versus the healthy liver tissue.
 4. The methodof claim 3, wherein the enzymes differentially active in the diseasedliver tissue comprise a set of enzymes wherein activity of the set hasbeen shown to correlate to a stage of the liver disease.
 5. The methodof claim 3, further comprising measuring quantities of the plurality ofdetected reporters.
 6. The method of claim 3, wherein each of theenzymes is a protease.
 7. The method of claim 3, further comprisingcorrelating quantities of the plurality of detected reporters to a rateof progression or regression of the liver disease.
 8. The method ofclaim 3, wherein each of the enzymes is a serine protease, a cysteineprotease, a threonine protease, an aspartic protease, or ametalloprotease.
 9. The method of claim 8, wherein the reporters arereleased via enzymatic cleavage.
 10. The method of claim 3, wherein theliver disease is non-alcoholic fatty liver disease (NAFLD), nonalcoholicsteatohepatitis (NASH), hepatocellular carcinoma (HCC), alcoholic liverdisease, genetic liver diseases, autoimmune liver diseases,toxicity-induced liver disease, or metastatic liver cancer.
 11. Themethod of claim 3, wherein the proteases are selected from the groupconsisting of FAP, MMP2, ADAMTS2, FURIN, MMP14, GZMB, PRSS8, MMP8,ADAM12, CTSS, CTSA, CTSZ, CASP1, ADAMTS12, CTSD, CTSW, MMP11, MMP12,GZMA, MMP23B, MMP7, ST14, MMP9, MMP15, ADAMDEC1, ADAMTS1, GZMK, KLK11,MMP19, PAPPA, CTSE, PCSK5, and PLAU.
 12. The method of claim 3, furthercomprising the step of determining a stage of NASH.
 13. The method ofclaim 1, wherein the activity sensor comprises a multi-arm polyethyleneglycol (PEG) scaffold and the reporters comprise polypeptides linked tothe PEG scaffold.
 14. A composition for screening or diagnosis of liverdisease, the composition comprising: an activity sensor; and a reporterreleasably attached to the activity sensor, wherein the reporter isreleased from the activity sensor in the liver in the presence of anenzyme that is differentially expressed in liver affected by a diseaseversus healthy liver.
 15. The composition of claim 14, wherein thereporter is released from the activity sensor via enzymatic cleavage.16. The composition of claim 15, further comprising a plurality of theactivity sensors, wherein each activity sensors has a plurality of thereporters releasably attached thereto, wherein the plurality of activitysensors are released by a plurality of enzymes that are differentiallyexpressed in liver affected by the disease.
 17. The composition of claim16, wherein the plurality of enzymes are selected from the groupconsisting of FAP, MMP2, ADAMTS2, FURIN, MMP14, GZMB, PRSS8, MMP8,ADAM12, CTSS, CTSA, CTSZ, CASP1, ADAMTS12, CTSD, CTSW, MMP11, MMP12,GZMA, MMP23B, MMP7, ST14, MMP9, MMP15, ADAMDEC1, ADAMTS1, GZMK, KLK11,MMP19, PAPPA, CTSE, PCSK5, and PLAU.
 18. The composition of claim 16,wherein the activity sensor comprises a molecular scaffold and thereporters comprise polypeptides linked to the scaffold.
 19. Thecomposition of claim 18, wherein the molecular scaffold comprises apolyethylene glycol (PEG) scaffold.
 20. The composition of claim 19,wherein PEG scaffold comprises a multi-arm PEG molecule of between about30 and about 50 kDa.